Multifunctional single chain glycoprotein hormones comprising three or more β subunits

ABSTRACT

Glycosylated or nonglycosylated molecules of the formula
 
β 1 -(linker 1 ) n     1   -β 2 -(linker 2 ) n     2   -β 3 -(linker 3 ) n     3   -α;  (1)
 
β 1 -(linker 1 ) n     1   -β 2 -(linker 2 ) n     2   -α-(linker 3 ) n     3   -β 3  ;  (2)
 
β 1 -(linker 1 ) n     1   -α-(linker 2 ) n     2   -β 2 -(linker 3 ) n     3   -β 3  ;  (3)
 
and
 
α-(linker 1 ) n     1   -β 1 -(linker 2 ) n     2   -β 2 -(linker 3 ) n     3   -β 3    (4)
         wherein α is the α subunit of a vertebrate glycoprotein hormone or a variant thereof;   each β is independently a glycoprotein β subunit or a variant thereof,   each “linker” is a hydrophilic, flexible spacer equivalent to a peptide containing 1–100 amino acid residues; and   each n is a 0 or 1;   said compound optionally comprising one or more additional β x (linker x ) n     x    and/or one or more additional α subunits are useful in protocols to enhance fertility in humans and in animals.

TECHNICAL FIELD

The invention relates to compositions that are useful in modulatingfertility in humans and animals. More specifically, it concerns singlechain forms of the glycoprotein hormones which contain multiple βsubunits of the hormones luteinizing hormone (LH) follicle stimulatinghormone (FSH), chorionic gonadotropin (CG), and/or thyroid stimulatinghormone (TSH) coupled to one or more of the α subunit.

BACKGROUND ART

In humans, four important glycoprotein hormone heterodimers (LH, FSH,TSH and CG) have differing β subunits but identical α subunits. Three ofthese hormones are directly related to fertility, and the fourth, TSH,is indirectly so related. Three of these hormones, LH, FSH and TSH, arepresent in virtually all other vertebrate species; CG has so far beenfound only in primates and in the placenta and urine of pregnant mares.

PCT application WO 90/09800, published 7 Sep. 1990 and incorporatedherein by reference, describes a number of modified forms of thesehormones. One important modification is a C-terminal extension of the βsubunit by the carboxy terminal peptide (CTP) of human chorionicgonadotropin or a variant thereof. CTP is the sequence of amino acidsextending from any one of positions 112–118 to position 145 of the βsubunit of human chorionic gonadotropin. PCT publication WO 94/24148published 27 Oct. 1994 and incorporated herein by reference describesmodifying these hormones by addition of the CTP at locations other thanthe C-terminus as well as variants of the CTP itself which are shorterthan the sequence defined above.

PCT publication WO 91/16922 published 14 Nov. 1991 describes a number ofchimeric and otherwise modified forms of the heterodimeric glycoproteinhormones. This disclosure is incorporated herein by reference as well.In addition, two PCT publications describe single chain forms of thesehormones wherein the α and β subunits are covalently linked to result ina fusion peptide of the formula β(linker)_(n)α, or α(linker)_(n)β,wherein n is 0 or 1 and α and β represent the respective subunits ofthese hormones. These publications, WO 95/22340 published 24 Aug. 1995and WO 96/05224 published 22 Feb. 1996 are incorporated herein byreference.

Forms of the single chain glycoprotein hormones in which the number ofcystine bridges has been depleted are disclosed in U.S. Pat. No.6,693,074 filed 19 Sep. 1997 and incorporated herein by reference.

PCT publication WO 99/25849 published 27 May 1999 and incorporatedherein by reference describes covalent single chain forms of theglycoprotein hormones which contain two β subunits. The two β subunitsmay be the same or different and may be coupled through linkers to eachother and to a common α subunit. These compounds are of the formulasβ¹-(linker¹)_(m)-α-(linker²)_(n)-β²;β¹-(linker¹)_(m)-β²-(linker²)_(n)-α; andα-(linker¹)_(m)-β¹-(linker²)_(n)-β².

the two β subunits are different, the single chain forms aremultifunctional.

In addition, PCT publication WO 00/23473 published 27 Apr. 2000 andincorporated herein by reference also describes modified forms of thesesingle chain hormones wherein one of the β subunits is coupledcovalently, optionally through a linker to the α subunit while a secondβ subunit is non-covalently associated with the single chain form.

U.S. Pat. No. 6,689,365 filed 5 May 2000 and incorporated herein byreference describes a subgenus of single chain forms of the formulaFSHβ-(linker¹)_(n) ₁ -LHβ(1−x)-(linker²)_(n) ₂ -α

-   -   wherein LHβ(1−x) refers to the LHβ subunit optionally containing        deletions of up to 7 amino acids from the carboxy terminus and        each linker is an amino acid sequence which is flexible and        hydrophilic; each n is 0 or 1. By adjusting the lengths of the        linkers, especially that between the LHβ subunit and α, the        ratio of FSH to LH activity can be fine-tuned.

Thus, while the art describes single chain multifunctional glycoproteinhormone forms, the multifunctionality has been limited to thatascribable to only two of the glycoprotein hormone heterodimers. It hasnow been found that single chain forms can be constructed wherein theactivity of at least three of the hormones can be exhibited based on asingle molecule. The relative degrees of these activities can beadjusted by controlling the spacing within the construct.

DISCLOSURE OF THE INVENTION

The compounds of the invention contain three or more β subunits, whichmay be the same or different, coupled to one or more α subunitsoptionally through linking moieties of variable lengths. Thus, thecompounds of the invention are of the formulas:β¹-(linker¹)_(n) ₁ -β²-(linker²)_(n) ₂ -β³-(linker³)_(n) ₃ -α  (1);β¹-(linker¹)_(n) ₁ -β²-(linker²)n ₂ -α-(linker³)_(n) ₃ -β³   (2);β¹-(linker¹)_(n) ₁ -α-(linker²)_(n) ₂ -β²-(linker³)_(n) ₃ -β³   (3);andα-(linker¹)_(n) ₁ -β¹-β¹-(linker²)_(n) ₂ -β²-(linker³)_(n) ₃ -β³  (4)

-   -   wherein α designates the α subunit common to the glycoprotein        hormones or a variant thereof, each β is independently the β        subunit of one of said glycoprotein hormones or a variant        thereof and each linker is independently a flexible hydrophilic        moiety that distances the coupled subunits by a spacing        equivalent to that generated by 1–100 amino acids, preferably        1–50 amino acids and each n is independently 0 or 1. In        addition, the above formulas may include one or two additional α        subunits along with an optional linker in positions which do not        link the α subunit to the α subunit already present, and may        also contain one or more additional β subunits linked at any        position. The α and β subunits or variants may be based on the        human forms or the forms derived from other vertebrates, such as        other primates, and preferably mammalian forms. The level of        activity of the various β subunits can be adjusted by adjusting        linker length.

Thus, in one aspect, the invention is directed to compounds of formulas(1)–(4); in other aspects, the invention is directed to recombinantmaterials for the production of these compounds and to methods for theirproduction as well as to methods for their use. In still other aspects,the invention is directed to antibodies which are specificallyimmunoreactive with the compounds of the invention and to formulationssuitable for pharmaceutical and veterinary use containing thesecompounds or the corresponding recombinant materials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a construct for the production ofTSHβ-CTP-FSHβ-CTP-CGβ-α.

FIG. 2 is a graph showing the ability of the product of the constructshown in FIG. 1 to bind the human chorionic gonadotropin (hCG) receptorin competition with labeled recombinant hCG.

FIG. 3 is a graph showing the ability of the construct shown in FIG. 1to bind FSH receptor.

FIG. 4 is a graph showing the ability of the construct of FIG. 1 toactivate the CG receptor.

FIG. 5 is a graph showing the ability of the construct shown in FIG. 1to activate the FSH receptor.

FIG. 6 is a graph showing the ability of the construct shown in FIG. 1to activate the TSH receptor.

MODES OF CARRYING OUT THE INVENTION

Four “glycoprotein” hormones in humans provide a family which includeshuman chorionic gonadotropin (hCG), follicle stimulating hormone (FSH),luteinizing hormone (LH), and thyroid stimulating hormone (TSH). All ofthese hormones are heterodimers comprised of a subunits which, for agiven species, are identical in amino acid sequence among the group, andβ subunits which differ according to each member of the family. Thus,normally these glycoprotein hormones occur as heterodimers composed of αand β subunits that are non-covalently associated. Most vertebratesproduce FSH, TSH and LH; chorionic gonadotropin has been found only inprimates, including humans, and in pregnant mares.

In animals, the α and β subunit of each hormone are encoded by differentgenes and are synthesized separately and then assembled into thenoncovalent heterodimeric complex. In the compounds of the invention atleast three β subunits are covalently linked to at least one α subunitto form a single-chain molecule which is essentially linear in primarystructure. The three dimensional structure conferred by secondary andtertiary structural considerations and energy of conformation isapparently sufficiently similar to the heterodimeric form to permit thefunctionality of the heterodimer represented by each of the β subunitsto be exhibited. The compounds of the present invention are particularlyadvantageous in the design of pharmaceuticals for inducing orcontrolling fertility since a multiplicity of hormone functions can beintroduced. Although LH, hCG and FSH are directly involved in thephysiological functions which control fertility, and thus are ofapparent use in this regard, it should be noted that TSH is an importantelement in such compositions since deficiencies in thyroid productionaffect ovulation.

The correlation between TSH and reproductive issues is well established.This has been reviewed, for example, in Mayer, et al., Thyroid andFertility (1998) 22:17–21. The correlation between thyroid conditionsand female reproductive conditions is discussed by Krassas, G. E., etal., Fertil. Steril. (2000) 74:2063–2070. The correlation in adult malerats is discussed by Jiang, J. Y., et al., Biol Repro. (2000)63:1637–1641. Further discussions of the correlation between thyroidfunction and human reproduction are found in articles by Vierhapper, H.,Hum. Repro. (1997) 12:2856; Moncayo, H. E., et al., ibid. 2854–2856.

In treating cells either in in vitro or in vivo for induction offertility, it is desirable to provide the effects of folliclestimulating hormone (FSH) as the major component. However, it isadvantageous to provide a typically lesser, but nevertheless significantamount of activity with respect for receptors for chorionicgonadotropin/luteinizing hormone. (The same receptor recognizes both CGand LH). The degree of LH activity that is desirable varies somewhatwith the particular subject or cells to be treated, but it is typicallysubstantially less than that of the FSH activity. TSH activity may alsobe required when the treatment is in vivo. By suitable manipulations ofthe components of the compounds of the invention, including the natureand length of the various optional linkers, the activity of the compoundwith respect to interaction with receptors for the various hormones canbe well controlled.

Thus, by adjusting the precise nature of the various components of thesingle chain compounds of the invention, the strength of the dosage andthe duration of the dosage of each individual component can be variedrelative to the remaining components while providing the opportunity toadminister all of these activities as a single dose. Such single dosageforms have considerable advantages in the practical application offertility or infertility treatments. For example, the dosage strength ofFSH could be increased by providing more than a single β subunit of FSHor by adjusting the spacing of the FSH from other components of thecompound. The duration of the dosage can be altered by altering theglycosylation state of the β subunits, each individually such that afinely tuned time course of administration of each component can beachieved by administration of a single compound.

The Subunit Components

As used herein, the common α subunit, and FSHβ, LHβ, and CGβ subunits aswell as the heterodimeric forms have their conventional definitions andrefer to the proteins having the amino acid sequences known in the artper se, or allelic variants thereof, regardless of the glycosylationpattern exhibited or other derivatization of the amino acid side chains.

“Native” forms of these peptides are those which have the amino acidsequences that have been isolated from the relevant vertebrate tissue,and have these known sequences per se, or those of their allelicvariants.

“Variant” forms of the subunits in the proteins and of CTP units (seebelow) are those which have deliberate alterations, includingtruncations, in amino acid sequences of the native protein produced by,for example, site-specific mutagenesis or by other recombinantmanipulations, or which are prepared synthetically.

These alterations consist of 1–7, preferably 1–5, more preferably 1–3,and even more preferably 1 amino acid changes, including deletions,and/or insertions, and/or substitutions, including in addition tonon-conservative substitutions, conservative amino acid substitutions.The resulting variants must retain an activity that reflects that thenative hormone—i.e., they must retain the biological activity of thenative hormone so as to behave as agonists.

“Conservative analog” means, in the conventional sense, an analogwherein the residue substituted is of the same general amino acidcategory as that for which substitution is made. Amino acids have beenclassified into such groups, as is understood in the art, by, forexample, Dayhoff, M., et al., Atlas of Protein Sequences and Structure(1972) 5:89–99. In general, acidic amino acids fall into one group;basic amino acids into another; neutral hydrophilic amino acids intoanother; and so forth. More specific classifications are set forth in WO96/05224 incorporated by reference above.

One set of preferred variants is that wherein the glycosylation sites ofeither the α or β subunits or both or of the CTP or partial CTP havebeen altered. Some useful variants of the hormone quartet describedherein are set forth in U.S. Pat. No. 5,177,193 issued 5 Jan. 1993 andincorporated herein by reference. The glycosylation patterns can bealtered by destroying the relevant sites, by adding one or more sites,or, in the alternative, by changing the host cell in which the proteinis produced.

Variants also include those with noncritical regions altered or removed.Such deletions and alterations may comprise entire loops, so thatsequences of considerably more than 10 amino acids may be deleted orchanged. The resulting variants must, however, retain at least thereceptor binding domains and the regions involved in signaltransduction.

There is considerable literature on variants of the glycoproteinhormones and it is clear that a large number of possible variants whichresult in agonist activity can be prepared. Such variants are disclosed,for example, in Chen, F., et al., Molec Endocrinol (1992) 6:914–919;Yoo, J., et al., J Biol Chem (1993) 268:13034–13042; Yoo, J., et al,. JBiol Chem (1991) 266:17741–17743; Puett, D., et al., GlycoproteinHormones, Lusbader, J. W., et al., EDS, Springer Verlag New York (1994)122–134; Kuetmann, H. T., et al., (ibid.) pages 103–117; Erickson, L.D., et al., Endocrinology (1990) 126:2555–2560; and Bielinska, M., etal., J Cell Biol (1990) 111:330a (Abstract 1844).

Other variants include those wherein one or more cystine-bonds aredeleted, typically by substituting a neutral amino acid for one or bothcysteines which participate in the link. Particularly preferred cystinebonds which may be deleted are those between positions 26 and 110 andbetween positions 23 and 72 in the human forms and correspondingpositions in other vertebrate forms.

As used herein “peptide” and “protein” are used interchangeably, sincethe length distinction between them is arbitrary.

“Noncritical” regions of the α and β subunits are those regions of themolecules not required for biological activity. In general, theseregions are distant from binding sites, precursor cleavage sites, andcatalytic regions. Regions critical for inducing proper folding, bindingto receptors, catalytic activity and the like should be evaluated. Itshould be noted that some of the regions which are critical in the caseof the dimer become noncritical in single chain forms since theconformational restriction imposed by the molecule may obviate thenecessity for these regions. The ascertainment of noncritical regions isreadily accomplished by deleting or modifying candidate regions andconducting an appropriate assay for the desired activity. Regions wheremodifications result in loss of activity are critical; regions whereinthe alteration results in the same or similar activity are considerednoncritical.

As used herein, the “CTP unit” refers to an amino acid sequence found atthe carboxy terminus of human chorionic gonadotropin β subunit whichextends from amino acid 112–118 to residue 145 at the C-terminus. Thus,each “complete” CTP unit contains 28–34 amino acids, depending on theN-terminus of the CTP.

By a “partial” CTP unit is meant an amino acid sequence which occursbetween positions 112–118 to 145 inclusive, but which has at least oneamino acid deleted from the shortest possible “complete” CTP unit (i.e.,from positions 118–145). These “partial” sequences are included in thedefinition of “variants” with respect to CTP. The “partial” CTP unitscontain at least one O-glycosylation site. The CTP unit contains fourglycosylation sites at the serine residues at positions 121 (site 1);127 (site 2); 132 (site 3); and 138 (site 4). The partial forms of CTPuseful in agonists will contain one or more of these sites arranged inthe order in which they appear in the native CTP sequence, althoughintervening sites may be omitted.

Preferred Compounds

In the compounds of formulas (1)–(4), each β subunit may be different,or all β subunits may be the same, or two may the same and the otherdifferent. Because CG and LH are recognized by the same receptor, inmany embodiments, the β subunits of these hormones are interchangeable.As CGβ automatically contains a CTP, when CGβ resides in the molecule,it is preferred that it be coupled to the downstream portion with ashorter linker or no linker at all as compared to the remaining βsubunits. Preferred embodiments of the linkers in general include CTPunits and variants thereof as defined above.

While the invention is illustrated by a tetramer which contains, inaddition to the common α subunit, β subunits from all three types ofactivities: FSH, TSH and CG/LH, additional α and β subunits can also beemployed. In a particularly preferred embodiment, all four β subunits,CGβ, LHβ, FSHβ and TSHβ are included. In this family, in embodimentswhere only one α subunit is present, the compounds are of the formβ¹(linker¹)_(n) ₁ -β²(linker²)_(n) ₂ -β³(linker³)_(n) ₃ -β⁴(linker⁴)_(n)₄ -α,  (5)β¹(linker¹)_(n) ₁ -β²(linker²)_(n) ₂ -β³(linker³)_(n) ₃-α-β⁴(linker⁴)_(n) ₄ ,  (6)β¹(linker¹)_(n) ₁ -β²(linker²)_(n) ₂ -α-β³(linker³)_(n) ₃-β⁴(linker⁴)_(n) ₄ ,  (7)β¹(linker¹)_(n) ₁ -α-β²(linker²)_(n) ₂ -β³(linker³)_(n) ₃-β⁴(linker⁴)_(n) ₄ ,  (8)α-β¹(linker¹)_(n) ₁ -β²(linker²)_(n) ₂ -β³(linker³)_(n) ₃-β⁴(linker⁴)_(n) ₄ .  (9)

Particularly preferred, among these, are embodiments wherein each of theβ subunits is of a different hormone—i.e., in any order, βCG, βLH, βTSHand βFSH.

In general, the linker moieties space the components of the moleculesderived from the glycoprotein hormones at distances equivalent to 1–100amino acids, preferably 1–50 amino acids. It is most preferred that thelinkers consist of a peptide of 1–50 gene-encoded amino acids so thatrecombinant production of the compounds of formulas (1)–(4) ispractical. The linkers may preferably comprise, as stated above, CTPunits or partial CTP units, but may also consist of sequences ofhydrophilic amino acids or amino acids lacking substantial side chains,such as glycine and serine. In general, the linkers should behydrophilic.

However, as it is possible to produce the compounds of formulas (1)–(4)synthetically, the linkers need not necessarily consist of naturallyoccurring amino acids but may include non-naturally occurringcounterparts, and may also include hydrophilic linkers in general, suchas polyethylene glycols or substituted polyesters or polyamides. Thesubunits derived from the glycoprotein hormones are preferably linked ina head-tail sequence, again permitting recombinant production of thesepeptides, but may also be linked head-head (i.e., through the N-termini,in this case requiring a linker) or tail-tail (again requiring alinker).

By adjusting the nature of the linkers, the glycosylation levels of thesingle chain multifunctional hormone can be adjusted. One advantage ofthe multiplicity of β subunits is also an inherently higher level ofglycosylation, which is believed to enhance half-life. Thus, thecompounds of the invention have the inherent advantage of longhalf-life.

Particularly preferred embodiments of the hormones of the inventioninclude, shown N-C:TSHβ-FSHβ-CTP-LHβ(1–114)-α;CGβ-LHβ-CTP-FSHβ-α;FSHβ-CTP-LHβ-TSHβ-α;FSHβ-LHβ-CGβ-α;TSHβ-CTP-FSHβ-CTP-CGβ-LHβ-CTP-α;TSHβ-α-LHβ-CTP-FSHβ;FSHβ-CTP-LHβ-CTP-CG-α;FSHβ-α-LHβ-CTP-CGβ;FSHβ-LHβ-α-CGβ-TSHβ;LHβ-CTP-TSHβ-CTP-α-CGβ;CGβ-FSHβ-CTP-LHβ-α-TSHβ-CTP;TSHβ-FSHβ-CTP-α-LHβ-CTP.

In all of the above, the CTP linkers maybe complete—i.e., 112–145,113–145 . . . 118–145, or partial.

While for human use, the human forms of the α and β subunits aredesirable, it should be noted that the corresponding forms in othervertebrates are useful in veterinary contexts. The FSH, TSH and LHsubunits from bovine, ovine, equine, porcine, feline, canine, and otherspecies are appropriate to indications affecting these species.

Other Modifications

The single-chain proteins of the invention may be further conjugated orderivatized in ways generally understood to modify amino acid sequences,such as phosphorylation, glycosylation (both N- and O- linked),deglycosylation of ordinarily glycosylated forms, acylation,modification of amino acid side chains (e.g., conversion of proline tohydroxyproline) and similar modifications analogous to thoseposttranslational events which have been found to occur generally.

The glycosylation status of the hormones of the invention isparticularly important. The hormones may be prepared in nonglycosylatedform either by producing them in prokaryotic hosts or by mutating theglycosylation sites normally present in the subunits and/or any CTPunits that may be present. Both nonglycosylated versions and partiallyglycosylated versions of the hormones can be prepared by manipulatingthe glycosylation sites. Normally, glycosylated versions are, of course,also included within the scope of the invention.

As is generally known in the art, the single-chain proteins of theinvention may also be coupled to labels, carriers, solid supports, andthe like, depending on the desired application. The labeled forms may beused to track their metabolic fate; suitable labels for this purposeinclude, especially, radioisotope labels such as iodine 131, technetium99, indium 111, and the like. The labels may also be used to mediatedetection of the single-chain proteins in assay systems; in thisinstance, radioisotopes may also be used as well as enzyme labels,fluorescent labels, chromogenic labels, and the like. The use of suchlabels permits localization of the relevant receptors since they can beused as targeting agents for such receptors.

The proteins of the invention may also be coupled to carriers to enhancetheir immunogenicity in the preparation of antibodies specificallyimmunoreactive with these new modified forms. Suitable carriers for thispurpose include keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) and diphtheria toxoid, and the like. Standard coupling techniquesfor linking the modified peptides of the invention to carriers,including the use of biofunctional linkers, can be employed.

Similar linking techniques, along with others, may be employed to couplethe proteins of the invention to solid supports. When coupled, theseproteins can then be used as affinity reagents for the separation ofdesired components with which specific reaction is exhibited. Thus, theyare useful in the purification and isolation of the receptors with whichthe appropriate β subunit interacts.

Preparation Methods

Methods to construct the proteins of the invention are well known in theart. The most practical approach at present is to synthesize thesematerials recombinantly by expression of the nucleotide sequenceencoding the desired protein. A nucleic acid containing the nucleotidesequence encoding the single-chain forms, including variants, can beprepared from native sequences, or synthesized de novo or usingcombinations of these techniques. Techniques for site-directedmutagenesis, ligation of additional sequences, amplification such as byPCR, and construction of suitable expression systems are all, by now,well known in the art. Portions or all of the DNA encoding the desiredprotein can be constructed synthetically using standard solid phasetechniques, preferably to include restriction sites for ease ofligation. Suitable control elements for transcription and translation ofthe included coding sequence can be provided to the DNA codingsequences. As is well known, expression systems are now availablecompatible with a wide variety of hosts, including prokaryotic hostssuch as E. coli or B. subtilis and eucaryotic hosts such as yeast, otherfungi such as Aspergillus and Neurospora, plant cells, insect cells,mammalian cells such as CHO cells, avian cells, and the like.

The choice of host is particularly pertinent to posttranslationalevents, most particularly including glycosylation. The location ofglycosylation is mostly controlled by the nature of the glycosylationsites within the molecule; however, the nature of the sugars occupyingthis site is also influenced by the nature of the host. Accordingly, afine-tuning of the properties of the hormones of the invention can beachieved by proper choice of host.

A particularly preferred form of gene for the α subunit portion, whetherthe α subunit is modified or unmodified, is the “minigene” construction.As used herein, the α subunit “minigene” refers to the gene constructiondisclosed in Matzuk, M. M., et al., Mol Endocrinol (1988) 2:95–100, inthe description of the construction of pM²/CG α or pM²/α.

For recombinant production, modified host cells using expression systemsare used and cultured to produce the desired protein. These terms areused herein as follows:

A “modified” recombinant host cell, i.e., a cell “modified to contain”the recombinant expression systems of the invention, refers to a hostcell which has been altered to contain this expression system by anyconvenient manner of introducing it, including transfection, viralinfection, and so forth. “Modified cells” refers to cells containingthis expression system whether the system is integrated into thechromosome or is extrachromosomal. The “modified cells” may either bestable with respect to inclusion of the expression system or theencoding sequence may be transiently expressed. In short, recombinanthost cells “modified” with the expression system of the invention refersto cells which include this expression system as a result of theirmanipulation to include it, when they natively do not, regardless of themanner of effecting this incorporation.

“Expression system” refers to a nucleic acid molecule which includes acoding nucleotide sequence to be expressed and those accompanyingcontrol sequences necessary to effect the expression of the codingsequence. Typically, these controls include a promoter, terminationregulating sequences, and, in some cases, an operator or other mechanismto regulate expression. The control sequences are those which aredesigned to be functional in a particular target recombinant host celland therefore the host cell must be chosen so as to be compatible withthe control sequences in the constructed expression system.

If secretion of the protein produced is desired, an additionalnucleotide sequence encoding a signal peptide is also included so as toproduce the signal peptide operably linked to the desired single-chainhormone to produce the preprotein. During translation, the signalpeptide is cleaved to release the mature single-chain hormone.

As used herein “cells,” “cell cultures,” and “cell lines” are usedinterchangeably without particular attention to nuances of meaning.Where the distinction between them is important, it will be clear fromthe context. Where any can be meant, all are intended to be included.

The protein produced may be recovered from the lysate of the cells ifproduced intracellularly, or from the medium if secreted. Techniques forrecovering recombinant proteins from cell cultures are well understoodin the art, and these proteins can be purified using known techniquessuch as chromatography, gel electrophoresis, selective precipitation,and the like.

All or a portion of the hormones of the invention may be synthesizeddirectly using peptide synthesis techniques known in the art andsynthesized portions may be ligated chemically or enzymatically.

Antibodies

The proteins of the invention may be used to generate antibodiesspecifically immunoreactive with these new compounds. These antibodiesare useful in a variety of diagnostic and therapeutic applications.

The antibodies are generally prepared using standard immunizationprotocols in mammals such as rabbits, mice, sheep or rats, and theantibodies are titered as polyclonal antisera to assure adequateimmunization. The polyclonal antisera can then be harvested as such foruse in, for example, immunoassays. Antibody-secreting cells from thehost, such as spleen cells, or peripheral blood leukocytes, may beimmortalized using known techniques and screened for production ofmonoclonal antibodies immunospecific with the proteins of the invention.“Antibodies” include any fragment which retains the requiredimmunospecificity, such as F_(ab), F_(ab′), F_((ab′)2), F_(v) and soforth. Thus, the antibodies may also be prepared using recombinanttechniques, typically by isolating nucleotide sequences encoding atleast the variable regions of monoclonal antibodies with the appropriatespecificity and constructing appropriate expression systems. Thisapproach permits any desired modification such as production of F_(v)forms, chimeric forms, “humanized” forms and the like.

By “immunospecific for the proteins of the invention” is meantantibodies which specifically bind the referent compound of theinvention, but not the heterodimers or any of the included subunits perse or any single-chain forms which include only a single or only two βsubunits, within the general parameters considered to determine affinityor nonaffinity. It is understood that specificity is a relative term,and an arbitrary limit could be chosen, such as a difference in specificbinding of 100-fold or greater. Thus, an immunospecific antibodyincluded within the invention is at least 100 times more reactive withthe specified protein than with the corresponding heterodimers, priorart single-chain forms or separate subunits. Such antibodies can beobtained, for example, by screening for those that bind the inventioncompounds and discarding those that also bind the heterodimers, subunitsor prior art single-chain forms.

The antibodies of the invention are particularly useful in assessing thelevels of the invention compounds in the body fluids of a subject. Thus,therapeutic protocols can be monitored conveniently using standardimmunoassays involving these antibodies or fragments as defined above.In addition, the antibodies are useful in purification of the singlechain compounds of the invention and in analysis for production of theseproteins in various contexts, including synthetic and recombinantsystems.

Formulation and Methods of Use

The proteins of the invention are formulated and administered usingmethods comparable to those known for the heterodimers corresponding tothem. Thus, formulation and administration methods will vary accordingto the particular hormone or hormone combination used. However, thedosage level and frequency of administration may be altered as comparedto the heterodimer, especially if CTP units are present in view of theextended biological half life due to its presence.

Formulations for proteins of the invention are those typical of proteinor peptide drugs such as found in Remington's Pharmaceutical Sciences,latest edition, Mack Publishing Company, Easton, Pa. Generally, proteinsare administered by injection, typically intravenous, intramuscular,subcutaneous, or intraperitoneal injection, or using formulations fortransmucosal or transdermal delivery. These formulations generallyinclude a detergent or penetrant such as bile salts, fusidic acids, andthe like. These formulations can be administered as aerosols orsuppositories or, in the case of transdermal administration, in the formof skin patches. Oral administration is also possible provided theformulation protects the peptides of the invention from degradation inthe digestive system.

Optimization of dosage regimen and formulation is conducted as a routinematter and as generally performed in the art. These formulations canalso be modified to include those suitable for veterinary use.

The compounds of the invention may be used in many ways, most evidentlyas substitutes for the heterodimeric forms of the hormones. Thus, likethe heterodimers, the agonist forms of the single-chain hormones of theinvention can be used in treatment of infertility, as aids in in vitrofertilization techniques, and other therapeutic methods associated withthe native hormones. These techniques are applicable to humans as wellas to other animals. The choice of the single-chain protein in terms ofits species derivation will, of course, depend on the subject to whichthe method is applied.

The invention compounds are also useful as reagents in a manner similarto that employed with respect to the heterodimers.

In addition, the compounds of the invention may be used as diagnostictools to detect the presence or absence of antibodies that bind to thenative proteins to the extent such antibodies bind to the relevantportions of these single chain compounds in biological samples. They arealso useful as control reagents in assay kits for assessing the levelsof these hormones in various samples. Protocols for assessing levels ofthe hormones themselves or of antibodies raised against them arestandard immunoassay protocols commonly known in the art. Variouscompetitive and direct assay methods can be used involving a variety oflabeling techniques including radio-isotope labeling, fluorescencelabeling, enzyme labeling and the like.

The compounds of the invention are also useful in detecting andpurifying receptors to which the native hormones bind. Thus, thecompounds of the invention may be coupled to solid supports and used inaffinity chromatographic preparation of receptors or antihormoneantibodies. The resulting receptors are themselves useful in assessinghormone activity for candidate drugs in screening tests for therapeuticand reagent candidates. Of course, account must be taken of thespecificity of the β subunits if the β subunits are different.

Finally, the antibodies uniquely reactive with the compounds of theinvention can be used as purification tools for isolation of thesematerials in their subsequent preparations. They can also be used tomonitor levels of these compounds administered as drugs.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Preparation of Invention Compounds

Expression vectors were prepared for production of the proteins andtheir secretion from Chinese hamster ovary (CHO) cells. The construct ofthe nucleotide sequence encoding the compound that was prepared andtested is shown in FIG. 1. This compound is TSHβ-CTP-FSHβ-CTP-CGβ-α.

In this construct, the various β subunits and α subunit are the humanforms. The expression system is constructed in a derivative of pM² avector originally described in Matzuk, M. M., et al., Mol Endocrinol(1988) 2:95–100. The derivative vector, pM²HA, contains a polylinkerwhich includes a number of unique cloning sites as described by Sachais,B., et al., J Biol. Chem. (1993) 268:2319–2323. This vector, containinga single chain construct comprising CGβ-α as shown in FIG. 1 isdescribed in Sugihara, T., et al., Proc. Natl. Acad. Sci. USA (1995)92:2041–2045. As shown in FIG. 1, the three exons of the CGβ codingsequence from positions 1–145 are directly fused to the exons whichconstitute the α subunit encoding sequence. The upstream FSHβ-CTPportion of the final construct to prepare the construct containing exons1, 2 and 3 of the FSHβ encoding sequence which is fused to thenucleotide sequence encoding positions 118–145 of CGβ as described byKanda, M., et al., Mol. Endocrinol. (1999) 13:1873–1881 and as shown inFIG. 1. The upstream TSHβ portion is then obtained from the constructdescribed by Fares, F., et al., Endocrinol. (1998) 139:2459–2464 toobtain the final construct depicted in FIG. 1.

The resulting vector was transfected into CHO cells and the cells werecultured to encourage expression. The tetramer, designated T2-7,containing the three β subunits and α subunit was secreted successfullyand its identity confirmed on Western blot using monoclonal antibodiesdirected to FSHβ, CGβ and TSHβ and by antibodies to human α. All ofthese antibodies bind to a secreted protein of the proper size.

EXAMPLE 2 Binding of the LH or FSH Receptor

The tetramer compound of Example 1 was tested for its ability to competefor binding to receptor with I-125 labeled hCG or FSH heterodimers orother constructs as appropriate. The procedures for assay are those setforth in Kanda, M., et al., Mol Endocrinol (1999) 13:1873–1881, citedabove and incorporated herein by reference.

Briefly, for assessing binding to the CG/LH receptor, CHO cellsexpressing human LH receptor (4×10⁵/tube) were incubated with one nglabeled CG in competition with increasing concentrations of unlabeled CGas a standard or with increasing amounts of the samples to be tested, at22° C. for 18 hours. The decrease in label in the presence of samplemeasures the binding ability in the sample. The results are shown inFIG. 2.

As shown, both T2.7 constructs show binding affinity comparable to thatof the CG heterodimer (solid diamonds). The tetramer construct (solidsquares) provides an EC₅₀ only approximately 5 fold more than the CGheterodimer. Several repetitions of this assay, however, show that thetetramer binds to the CG receptor with an EC₅₀ of only two-three foldhigher than the heterodimer.

A comparable essay testing ability of the compounds to bind the FSHreceptor displayed on CHO cells in competition with I-125 labeled FSH,in a protocol otherwise identical to that set forth above, was alsoperformed. These results are shown in FIG. 3. Again, the tetramer to theFSH receptor shows only slightly less affinity to receptor than therecombinantly produced heterodimer or the single chain trimer, FSH-CG-α.

EXAMPLE 3 Agonist Activity With Respect to LH, FSH and TSH

The tetramer of Example 1 was tested for ability to stimulate cyclic AMPproduction in CHO cells that display the CG/LH receptor, the FSHreceptor, or the TSH receptor. The procedure is that of Kanda, et al.,(1999) cited above. Briefly, the total extracellular and intracellularamount of cAMP is determined using the Adenyl Cyclase Activation FlashPlate Kit (NEN Life Science Products, Boston Mass.) as per themanufacturer's instructions. CHO cells (5×10⁴ cells per well) expressingeither LH/CG, FSH or TSH receptor were incubated with tetramer orcontrol for 2 hours at room temperature. cAMP labeled with I-125 wasadded and the cells were incubated for an additional 16–18 hours at roomtemperature. The flash plates were read in a Packard Top gamma counterand each experiment was performed 2–3 times. The cAMP content isexpressed in pmol/ml.

The results for the three β subunit component related receptors areshown in FIGS. 4–6.

FIG. 4 shows the results when the tetramer T.7 was tested in comparisonwith CG heterodimer. The ability of each to stimulate cAMP production iscomparable.

FIG. 5 shows the results when the test is performed with respect to theFSH receptor. The tetramer (solid squares) shows stimulating activitycomparable both to recombinant FSH (solid diamonds) and as compared tothe single chain FSH-CTP-CG-α (solid circles).

Similar results were obtained with regard to TSH receptor agonistactivity. As shown in FIG. 6, tetramer 2.7 (solid squares) is onlyslightly less agonistic than the TSH dimer (solid circles) or TSHobtained from NIH (solid diamonds); all of these compounds were moreeffective agonists than the single chain form TSH-CTP-α (opentriangles).

1. A glycosylated or nonglycosylated proteinaceous compound havingagonist activity for at least one glycoprotein hormone, and having astructure selected from the group consisting ofβ¹-(linker¹)_(n) ₁ -β²(linker²)_(n) ₂ -β³-(linker³)_(n) ₃ -α  (1);β¹-(linker¹)_(n) ₁ -β²-(linker²)_(n) ₂ -α-(linker³)_(n) ₃ -β³   (2);β¹-(linker¹)_(n) ₁ -α-(linker²)_(n) ₂ -β²-(linker³)_(n) ₃ -β³   (3); andα-(linker¹)_(n) ₁ -β¹-(linker²)_(n) ₂ -β²-(linker³)_(n) ₃ -β³   (4)wherein α is the α subunit of a vertebrate glycoprotein hormone or avariant thereof that binds to the corresponding glyco protein hormonereceptor; each β is independently a glycoprotein β subunit or a variantthereof; each “linker” is a hydrophilic, flexible spacer equivalent to apeptide containing 1–100 amino acid residues; and each n is a 0 or 1;said compound optionally comprising one or more additionalβ^(x)(linker^(x))_(n) _(x) and/or one or more additional α glycoproteinhormone subunits.
 2. The compound of claim 1 which is of the formulaβ¹-(linker¹)_(n) ₁ -β²-(linker²)_(n) ₂ -β³-(linker³)_(n) ₃ -α;  (1)β¹-(linker¹)_(n) ₁ -β²-(linker²)_(n) ₂ -α-(linker³)_(n) ₃ -β³;  (2)β¹-(linker¹)_(n) ₁ -α-(linker²)_(n) ₂ -β²-(linker³)_(n) ₃ -β³;  (3)α-(linker¹)_(n) ₁ -β¹-(linker²)_(n) ₂ -β²-(linker³)_(n) ₃ -β³;  (4)β¹(linker¹)_(n) ₁ -β²(linker²)_(n) ₂ -β³(linker³)_(n) ₃ -β⁴(linker⁴)_(n)₄ -α;  (5)β¹(linker¹)_(n) ₁ -β²(linker²)_(n) ₂ -β³(linker³)_(n) ₃-α-β⁴(linker⁴)_(n) ₄ ;  (6)β¹(linker¹)_(n) ₁ -β²(linker²)_(n) ₂ -α-β³(linker³)_(n) ₃-β⁴(linker⁴)_(n) ₄ ;  (7)β¹(linker¹)_(n) ₁ -α-β²(linker²)_(n) ₂ -β³(linker³)_(n) ₃-β⁴(linker⁴)_(n) ₄ ; or  (8)α-β¹(linker¹)_(n) ₁ -β²(linker²)_(n) ₂ -β³(linker³)_(n) ₃-β⁴(linker⁴)_(n) ₄ .  (9)
 3. The compound of claim 1 or 2 wherein each βis different.
 4. The compound of claim 1 or 2 wherein at least onelinker is independently a complete or partial CTP comprising at leastone glycosylation site or a variant thereof, wherein CTP refers to theamino acid sequence at positions 112–118 to 145 of human chorionicgonadotropin β subunit.
 5. The compound of claim 1 or 2 wherein saidprotein consists of naturally occurring amino acids.
 6. The compound ofclaim 1 or 2 wherein each β and α subunit is human native subunit. 7.The compound of claim 1 which is of formula (1).
 8. The compound ofclaim 7 which is TSHβ-CTP-FSHβ-CTP-CGβ-α.
 9. The compound of claim 2which is of formula (5).
 10. The compound of claim 9 wherein each βsubunit is different.
 11. The compound of claim 1 or 2 coupled to asolid support.